西双版纳黄瓜β-胡萝卜素生物合成相关基因的克隆及表达分析
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摘要
西双版纳黄瓜是我国特有的黄瓜变种资源，达到生理成熟度的西双版纳黄瓜老瓜的果肉和胎座均为橙黄色，该特殊性状的形成主要是由于-胡萝卜素的大量积累所致。-胡萝卜素是一类重要的类胡萝卜素，对人类的健康十分有益。本研究以西双版纳黄瓜为主要研究对象，基于黄瓜全基因组测序结果，结合前人对类胡萝卜素生物合成酶基因的研究，克隆黄瓜中-胡萝卜素生物合成相关酶基因，并进行序列分析，以了解不同酶基因的结构特点。与已公布的栽培黄瓜变种“9903”和“GY14”的参考序列进行比较分析，检测不同变种间相关酶基因在核苷酸水平的差异。结合113份黄瓜的重测序结果，筛选西双版纳黄瓜中的特异位点，开发相关分子标记。利用实时荧光定量PCR技术，对-胡萝卜素生物合成相关的5个酶基因在表达水平进行相对定量分析，以比较西双版纳黄瓜“BN35”和栽培变种“JD7”实不同发育时期的基因表达量的变化，上述研究结果将为深入探讨西双版纳黄瓜-胡萝卜素积累的分子机制，同时也为进一步挖掘黄瓜优异基因，改良瓜类作物的营养品质奠定基础。主要研究结果如下：
1.利用特异引物PCR技术，分别克隆获得-胡萝卜素生物合成途径中的重要酶基因CsPsy1、CsZds、CsLcyB、CsChxB的cDNA序列和CsPsy1、CsLcyB、CsChxB的DNA序列。对基因编码氨基酸序列进行基本理化及结构分析。结果显示，CsPSY1、CsZDS、CsCHXB均含叶绿体预测导肽，CsCHXB预测有3个跨膜构，推测其为膜结合蛋白或与膜结合才能发挥作用。特征序列分析结果显示，CsPSY1有两个特征序列，CsZDS、CsLCYB均含有NADB-Rossmann superfamily特征结构，CsCHXB含多个组氨酸受体，且与脂肪酸羟化酶特征序列高度一致。4个基因的保守结构域基因编码氨基酸序列十分保守，与其他物种同源性很高，含有各自蛋白的特征序列，系统进化树分析表明与葫芦科物种进化关系较为相近。
2.将克隆的上述基因与栽培黄瓜变种“9903”和“GY14”的参考序列进行比对，发现CsPsy1基因存在5个特异变异，包括4个SNP和1个InDel。CsLcyB基因编码区中存在1个SNP，该碱基的改变引起相应氨基酸的改变。CsChxB基因在第六内含子存在6个碱基T的插入，在编码区存在1个SNP，该碱基的变化引起相应氨基酸的改变。CsZds基因的cDNA无碱基变化。对113份黄瓜重测序材料的序列进行比对，发现CsChxB基因的SNP位点在西双版纳黄瓜中的特异性为100%，在94份普通黄瓜中均不存在该变异。CsLcyB基因的SNP位点在西双版纳黄瓜中表现不特异。
3.针对-胡萝卜素生物合成相关的5个重要酶基因（CsPsy1、CsPds、CsZds、CsLcyB、CsChxB），比较分析西双版纳黄瓜和普通黄瓜在果实不同发育时期的表达量，结果表明，在果实发育过程中，西双版纳黄瓜5个基因的表达量呈先上升后下降的趋势，基本在转色期达到最高值，而普通黄瓜各基因的上调表达时期明显滞后。比较上述基因在表达水平的差异，推测CsZds、CsLcyB和CsChxB基因对西双版纳黄瓜果实-胡萝卜素的积累有较大影响。
4.优化了黄瓜PCR-SSCP分子标记方法，根据CsChxB基因特异SNP位点，筛选获得较好的引物SNPChxb1-178，能够通过聚丙烯酰胺凝胶电泳对西双版纳黄瓜和普通黄瓜进行有效区分。
Xishuangbanna cucumber (Cucumis sativus L.var. xishuangbannanesis Qi et Yuan) is a specialvariation germplasm only found in China until now. Orange flesh and placenta of mature fruit wereobviously different from cultivated cucumbers.-carotene content of flesh in Xishuangbanna cucumberwas nearly one hundred times than that of common cucumbers, which was induced by the accumulationof-carotene.-carotene was an important kind of carotenoid and essential for our health. In this study,main genes in the-carotenoid pathway were focused on and key genes were cloned based on reportedresearches and related information of Cucumber Genome Database. Compared with the referencesequences of9930and GY14, specific sequences in Xishuangbanna cucumber were selected.Compared with the resequence database of113cucumber germplasm, the specific sequences relatedwith-carotene biosynthesis in Xishuangbanna cucumber were determined further and new molecularmarker was developed preliminarily. In order to evaluate the correlation of carotene accumulation withtranscriptional regulation of carotenoid biosynthesis related genes, using real-time PCR technique, theexpression of5genes during fruit development were studied in BN35(var. xishuangbannanesis) andJD7(var. sativus) respectively. Above results were benefit to understand the molecular mechanism ofcarotenoid accumulation, explicate elite genes of Xishuangbanna cucumber and improve qualitybreeding in cucumber. Main results were as followed:
The main results were as followed:
1. Four cDNA sequences encoding CsPsy1, CsZds, CsLcyB, CsChxB in-carotenoid biosynthesispathway and only three DNA sequences encoding CsPsy1, CsLcyB, CsChxB were cloned fromXishuangbanna cucumber. The sequence analyses showed that CsPSY1、CsZDS、CsCHXB containchloroplast leading peptide. There were three predicted transmembrane profiles for the deduced acidsequences of CsCHXB, while others’ weren’t detected. Conserved sequence analysis showed thatCsPSY1had two characteristic sequences, CsZDS and CsLCYB contained NADB-Rossmannsuperfamily sequences, and CsCHXB had a L-histidine receptor. The result of mutiple sequencealignment indicated that these genes were very conservative and shared high sequence identity withother orthologous genes. Above related enzymes had their own characteristic sequences. Phylogeneticanalysis showed predicted amino acids in cucumber were closest to cucurbitaceae.
2. Compared with9930and GY14, CsPsy1gene showed five differences which were4SNPand a13bp InDel. CsLcyB gene showed a SNP site within coding region, which brought about thechange of amino acid. CsChxB gene showed a TTTTTT insert region in the sixth intron and a SNP sitein coding region, which brought about the change of amino acid. CsZds gene showed no variationdefinitely. Blast the genome resequence data of113germplasm in cucumber, the SNP of CsChxB was100%specific in Xishuangbanna cucumber.
3. The expression level of CsPsy1, CsPds, CsZds, CsLcyB and CsChxB genes at different stagesduring fruit development were studied. The result of real-time PCR analysis showed that the expressionof-carotene biosynthesis relative genes of BN35were first increased and then decreased during fruit development, and the highest expression was at color turning period. The up regulation expression ofJD7was later than that of BN35. It was showed to some extent that CsZds, CsLcyB, CsChxB wererelated to-carotene accumulation.
4. The method of PCR-SSCP was optimized. According to the specific SNP of CsChxB gene,primer SNPChxb1-178was screened effectively while Xishuangbanna cucumber and cultivatedcucumber could be distinguished well.
1.利用特异引物PCR技术，分别克隆获得-胡萝卜素生物合成途径中的重要酶基因CsPsy1、CsZds、CsLcyB、CsChxB的cDNA序列和CsPsy1、CsLcyB、CsChxB的DNA序列。对基因编码氨基酸序列进行基本理化及结构分析。结果显示，CsPSY1、CsZDS、CsCHXB均含叶绿体预测导肽，CsCHXB预测有3个跨膜构，推测其为膜结合蛋白或与膜结合才能发挥作用。特征序列分析结果显示，CsPSY1有两个特征序列，CsZDS、CsLCYB均含有NADB-Rossmann superfamily特征结构，CsCHXB含多个组氨酸受体，且与脂肪酸羟化酶特征序列高度一致。4个基因的保守结构域基因编码氨基酸序列十分保守，与其他物种同源性很高，含有各自蛋白的特征序列，系统进化树分析表明与葫芦科物种进化关系较为相近。
2.将克隆的上述基因与栽培黄瓜变种“9903”和“GY14”的参考序列进行比对，发现CsPsy1基因存在5个特异变异，包括4个SNP和1个InDel。CsLcyB基因编码区中存在1个SNP，该碱基的改变引起相应氨基酸的改变。CsChxB基因在第六内含子存在6个碱基T的插入，在编码区存在1个SNP，该碱基的变化引起相应氨基酸的改变。CsZds基因的cDNA无碱基变化。对113份黄瓜重测序材料的序列进行比对，发现CsChxB基因的SNP位点在西双版纳黄瓜中的特异性为100%，在94份普通黄瓜中均不存在该变异。CsLcyB基因的SNP位点在西双版纳黄瓜中表现不特异。
3.针对-胡萝卜素生物合成相关的5个重要酶基因（CsPsy1、CsPds、CsZds、CsLcyB、CsChxB），比较分析西双版纳黄瓜和普通黄瓜在果实不同发育时期的表达量，结果表明，在果实发育过程中，西双版纳黄瓜5个基因的表达量呈先上升后下降的趋势，基本在转色期达到最高值，而普通黄瓜各基因的上调表达时期明显滞后。比较上述基因在表达水平的差异，推测CsZds、CsLcyB和CsChxB基因对西双版纳黄瓜果实-胡萝卜素的积累有较大影响。
4.优化了黄瓜PCR-SSCP分子标记方法，根据CsChxB基因特异SNP位点，筛选获得较好的引物SNPChxb1-178，能够通过聚丙烯酰胺凝胶电泳对西双版纳黄瓜和普通黄瓜进行有效区分。
Xishuangbanna cucumber (Cucumis sativus L.var. xishuangbannanesis Qi et Yuan) is a specialvariation germplasm only found in China until now. Orange flesh and placenta of mature fruit wereobviously different from cultivated cucumbers.-carotene content of flesh in Xishuangbanna cucumberwas nearly one hundred times than that of common cucumbers, which was induced by the accumulationof-carotene.-carotene was an important kind of carotenoid and essential for our health. In this study,main genes in the-carotenoid pathway were focused on and key genes were cloned based on reportedresearches and related information of Cucumber Genome Database. Compared with the referencesequences of9930and GY14, specific sequences in Xishuangbanna cucumber were selected.Compared with the resequence database of113cucumber germplasm, the specific sequences relatedwith-carotene biosynthesis in Xishuangbanna cucumber were determined further and new molecularmarker was developed preliminarily. In order to evaluate the correlation of carotene accumulation withtranscriptional regulation of carotenoid biosynthesis related genes, using real-time PCR technique, theexpression of5genes during fruit development were studied in BN35(var. xishuangbannanesis) andJD7(var. sativus) respectively. Above results were benefit to understand the molecular mechanism ofcarotenoid accumulation, explicate elite genes of Xishuangbanna cucumber and improve qualitybreeding in cucumber. Main results were as followed:
The main results were as followed:
1. Four cDNA sequences encoding CsPsy1, CsZds, CsLcyB, CsChxB in-carotenoid biosynthesispathway and only three DNA sequences encoding CsPsy1, CsLcyB, CsChxB were cloned fromXishuangbanna cucumber. The sequence analyses showed that CsPSY1、CsZDS、CsCHXB containchloroplast leading peptide. There were three predicted transmembrane profiles for the deduced acidsequences of CsCHXB, while others’ weren’t detected. Conserved sequence analysis showed thatCsPSY1had two characteristic sequences, CsZDS and CsLCYB contained NADB-Rossmannsuperfamily sequences, and CsCHXB had a L-histidine receptor. The result of mutiple sequencealignment indicated that these genes were very conservative and shared high sequence identity withother orthologous genes. Above related enzymes had their own characteristic sequences. Phylogeneticanalysis showed predicted amino acids in cucumber were closest to cucurbitaceae.
2. Compared with9930and GY14, CsPsy1gene showed five differences which were4SNPand a13bp InDel. CsLcyB gene showed a SNP site within coding region, which brought about thechange of amino acid. CsChxB gene showed a TTTTTT insert region in the sixth intron and a SNP sitein coding region, which brought about the change of amino acid. CsZds gene showed no variationdefinitely. Blast the genome resequence data of113germplasm in cucumber, the SNP of CsChxB was100%specific in Xishuangbanna cucumber.
3. The expression level of CsPsy1, CsPds, CsZds, CsLcyB and CsChxB genes at different stagesduring fruit development were studied. The result of real-time PCR analysis showed that the expressionof-carotene biosynthesis relative genes of BN35were first increased and then decreased during fruit development, and the highest expression was at color turning period. The up regulation expression ofJD7was later than that of BN35. It was showed to some extent that CsZds, CsLcyB, CsChxB wererelated to-carotene accumulation.
4. The method of PCR-SSCP was optimized. According to the specific SNP of CsChxB gene,primer SNPChxb1-178was screened effectively while Xishuangbanna cucumber and cultivatedcucumber could be distinguished well.
引文
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